The overall objective for this project is to develop and implement a comprehensive set of technologies to improve screening and diagnosis of conditions associated with Fragile X Syndrome (FXS). FXS is caused by an expansion of a cytosine-guanine-guanine (CGG) triplet repeat in the 5'-untranslated region of the FMR1 gene. FXS affects 1/4000 men and 1/6000 women. Full expansion to greater than 200 repeats is associated with hypermethylation of the FMR1 gene and complete loss of FMR1 protein production. A more modest expansion of the CGG repeats is associated with Fragile X-associated Tremor/Ataxia Syndrome (FX-TAS) in older men and primary ovarian insufficiency (FX-POI) in women. Recently published guidelines suggest follow-up chromosome and Fragile X testing for a diagnosis of autism spectrum disorder (ASD), which impacts 1/150 individuals-roughly 33 times the population incidence of Fragile X. Furthermore, promising drugs are currently in clinical trials and will require accurate and early identification of Fragile X patients. Improvements in testing for Fragile X will have important implications for a broad range of individuals of all ages across multiple mental and health conditions associated with this disorder. The current diagnostic approach for Fragile X testing is to perform PCR, followed by Southern blot as necessary, to determine the CGG repeat number and FMR1 methylation state. However, a major limitation of PCR is the inability to amplify full mutations and even many pre-mutation alleles. All current PCR methods lack the ability to resolve homozygosity. As a result, nearly 50% of clinical laboratories currently reflex all samples to the more expensive, laborious, and low-throughput Southern blot assay. Therefore, despite strong arguments for population-based screening for FXS (either newborn and/or carrier screening), the inability to reliably amplify full mutation repeat expansions makes wide-spread screening too expensive and inaccurate for implementation. Asuragen has undertaken an effort to improve the amplification of long GC-rich repeats in order to better serve the important clinical goals of robust diagnosis and screening for Fragile X mutations. In our preliminary data, we now demonstrate PCR amplification of up to at least ~1000 CGG repeats, the longest repeat length that is commercially available or published. This capability is the foundation for a PCR-based assay that can report both premutation and full mutation alleles and dramatically reduce or eliminate the number of cases reflexed to Southern blots. In this phase I SBIR project, we will establish efficient, automatable testing, develop a PCR_based test to resolve zygosity in female samples, and develop a PCR-based method for determining methylation status of the FMR1 gene. This will result in a diagnostic workflow that can support routine screening, and thus enable earlier interventions and improved treatment options for patients. PUBLIC HEALTH RELEVANCE: The long-term goal for this project is to improve screening and diagnosis of Fragile X Syndrome and related conditions. In this project, we will leverage our breakthrough in PCR of the FMR1 gene to develop a set of robust and accurate tests that are cost-effective and efficient. This will enable widespread screening to identify carriers and permit earlier diagnosis and intervention for Fragile X Syndrome patients.